genome res. 1992 yourno 60 5

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10.1101/gr.2.1.60Access the most recent version at doi:1992 2: 60-65Genome Res.

J Yourno

A method for nested PCR with single closed reaction tubes.

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A M et ho d for Nested PCR w i th SingleClosed Reaction TubesJoseph Yourno

W ad swo r t h C en t e r f o r L abora t o r i e s and Resea rch , New York S t at e Depar t men t o f Hea l t h , A l bany , New York 12201-0509

Toward the goal of reducing diagnos-tic false positives while retaininghigh sensitivity, a closed-tube nestedPCR procedure has been developedfor detecting low-copy-num ber hu-man immunodeflciency virus (HIV)ga g target DNA sequences. Mastermix for ampli f icat ion 2, in a hanginggel matrix at the reaction tube top,remains sequestered from the reac-t ion space of the tube during ampli -fication 1. A severalfold excess of in-ner over outer primers is bui l t intothe procedure to assure the high sen-sitivity of nested PCR. The master mixfor ampli f icat ion 2 is then intro-duced into the reaction space by cen-tr i fugation, and the second ampli f i -cation is performed as usual. Theclosed-tube nested procedure showssensit iv i ty approaching that of theopen-tube control procedure, whichdetects a single copy of HIV ga g tar-get DNA at near-theoretical fre-quency, typical ly with microgramyields of specific amplification prod-uc t .

N e s t e d P CR g r e a tl y e n h a n c e s s e n s it i v -i t y o f de t ec t i on o f ta rge t nu c l e i c ac i d se -quences . (1) T he appro ach i s based on ,f ir s t, t he am pl i f i ca t i on o f an ex t end ed se -q u e n c e a n d , s e c o n d , t h e a m p l i f i c a t i o n o fa n i n t e r n a l s e q u e n c e f r o m t h e p r o d u c tof t he f i rs t . T he e nh an ced sens i t i v i t y o ft h e m e t h o d i s a c h i e v e d b y c o n t r o l l i n gr e a c t i o n c o n d i t i o n s f o r e a c h a m p l i f i c a -t i on t o f avor genera t i o n o f t he des i r edproduc t . I n add i t i on t o t he usua l cons i d -e r a t i ons , mos t approaches r equ i r e a s ev-e r a l f o l d excess o f i nne r ove r ou t e r p r i m-e rs i n t h e s e c o n d a m p l i f i c a t i o n f ors a t is f a c to r y r e s u l t s . C o n v e n t i o n a l m e t h -o d s a c c o m p l i s h th i s b y a m p l i f y i n g o n l ya smal l a l i quo t o f t he compl e t ed f i r s ta m p l i f i c a t i o n m i x t u r e a f te r t r a n s fe r t o an e w r e a c t i o n t u b e f o r t h e s e c o n d a m p l i -f i ca t i on . T he i nc r ease d sens i t i v i t y o fnes t ed P CR i s bought a t t he p r i ce o f po-t en t i a l f a l s e pos it i ves , because t ube s con-t a i n i n g h i g h c o n c e n t r a t i o n s o f t h e f i r sta m p l i f i c a t i o n p r o d u c t m u s t b e o p e n e da n d m a n i p u l a t e d t o s e t u p t h e s e c o n da m p l i f i c a t i o n . A m e t h o d f o r e ff i c i e n tn e s t e d P C R t h a t e l i m i n a t e s t h e n e e d f o rt h e s e m a n i p u l a t i o n s w o u l d r e d u c e t h echa nce fo r fa l s e pos i t ives . T he app roachd e s c r i b e d h e r e i n i s b a se d o n t h e u s e o f as i n g l e re a c t i o n t u b e t h a t r e m a i n s c l o s e dfor bo t h amp l i f i ca t i on s a f t e r r eagen t s a r ei n t roduced fo r each . Reagen t s fo r t hes e c o n d a m p l i f i c a t i o n a r e s e q u e s t e re da n d p r e s e r v e d i n t h e r e a c t i o n t u b e d u r -i n g t h e f ir s t a m p l i f i c a t i o n , a n d t h e n i n -t r o d u c e d i n t o t h e r e a c t i o n m i x t u r e f o rt h e s e c o n d a m p l i f i c a t i o n . A c l o s e d -t u b en e s t e d p r o c e d u r e f o r d e t e c t i o n o f h u -m a n i m m u n o d e f i c i e n c y vi ru s ( H IV ) gags e q u e n c e s s h o w s s e n s i ti v i ty a p p r o a c h i n gt h e r a n g e o f t h e c o n v e n t i o n a l p r o c e -dure , wh i ch de t ec t s a s i ng l e t a rge t DNAsequence wi t h s t rong s i gna l a t nea r - t he -ore t i ca l f r equency .

MATERIALS AND METHODSStrategyP CR a m p l i f i c a t i o n 1 w i t h t e m p l a t e D N Ai s se t up i n t he usua l f a sh i on i n r eac t i ont u b e s a n d o v e r l a i d w i t h m i n e r a l o i l .O u t e r p r i m e r s a r e u s e d a t r e d u c e d c o n -c e n t r a t i o n . M a s t e r m i x f o r a m p l i f i c a t i o n2 is p r epa re d i n a me l t ed , t h i n aga roseg e l m a t r i x a n d i n t r o d u c e d i n t o t h e l i d -l o c k c h a m b e r o f t h e r e a c t i o n t u b e s . T h i sm i x c o n t a i n s i n n e r p r i m e r s a t s e v e r a l -fo l d excess ove r ou t e r p r i mer s fo r ampl i -f i ca t i on 1 . T he t u be deadsp ace i s f il l edw i t h m i n e r a l o i l t o j u s t b e l o w t h e l i d -l o c k l i n e b e fo r e t u b e c l o su r e . W h i l e t h ef ir s t a m p l i f i c a t i o n is r u n , t h e m a s t e r m i xf o r t h e s e c o n d r e m a i n s s e q u e s t e r e d a tt h e t u b e t o p , p r o t e c t e d f r o m e x t r e m et e m p e r a t u r e c h a n g e s t o w h i c h t h e l o w e rend of t he t ube i s sub j ec t ed . Af t e r t hef i rs t am pl i f i c a t i on i s com pl e t ed , t he r e-a g e n t s f o r t h e s e c o n d a m p l i f i c a t i o n a rei n t r o d u c e d b y b r i e f c e n t r i f u g a t i o n i n t ot h e r e a c t i o n s p a c e . T h e s e c o n d a m p l i f i -c a t i o n i s t h e n r u n a s u s u a l .

Standard DNA PreparationsP e r ip h e r a l b l o o d m o n o n u c l e a r c e ll s( P B M C ) f r o m n o r m a l d o n o r s ( A m e r i c a nRed Cross ) were i so l a t ed on F i co l l -Hy-p a q u e c u s h i o n s , a l i q u o t e d , a n d s t o r e d i nl i q u i d n i t ro g e n . D N A w a s p r e p a r e d f r o mt h a w e d , w a s h e d P B M C in P C R b u f fe rw i t h n o n i o n i c d e t e r g e n t s a n d p r o t e a s e Kb y t h e m e t h o d o f H i g u c h i . (2) D N A f r o mce l l s o f t he 8E 5 ce l l l i ne , whi ch con t a i no n e i n t e g r a t e d p r o v i r u s p e r g e n o m e , (3was i so l a t ed s i mi l a r l y . W h i t e ce l l coun t son P BM C and 8E 5 ce l l s were do ne by he -m a c y t o m e t e r . T h r e e f o l d d i l u t i o n s o f 8 E5D N A i n t o n o r m a l P M B C D N A w e r e m a d et o e s ta b l i s h s t a n d a r d s c o n t a i n i n g f r o m33 cop i es o f HIV prov i rus t o 0 cop i es i n

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0.5 i~g total DNA pe r 4 0 p.1 PCR buf fer.DNA con cen t r a t i ons a r e based on 1 I~gDNA per 200 ,000 ce l l s. S t andard p r epa ra -t i ons i n 1 .5 -ml gaske t ed sc r ew cap t ubes(S ars t ed t ) were bo i l ed , sna p-coo l ed i n i cew a t e r , a l i q u o t e d i n t o t h e s a m e t y p e o ft ube , and s t o r ed a t - 20~

PCR Amplificatio nA n e s t e d P C R p r o c e d u r e h a s b e e n d e -vised base d on SK38 and SK39 (4) as in-t e rna l p r i mer s fo r HIV ga g sequences . Re-ac t i on mi x pa ramet e r s a r e a s fo l l ows .Am pl i f i ca t i on 1 : Vol ume 50 I~1 , M gCI22 .5 r aM , ou t e r p r i m er s (HP L C-pur i f led ,Operon) 12 .5 pmol es each (S K3805'-GAG AAC CAA GGG GAA GTG ACATAG CAG G, 28-mer , HIV ga g (HXB2)687-712; SK390 5 ' -TAG AAC CGG TCTACA TAG TCT CTA AAG GG, 29-m er , HIVga g (HXB2) 874-903) , Am pl i T aq 0 .75u n i t , d e o x y n u c l e o s i d e t r i p h o s p h a t e s 2 0 0[.I.M each. Am pl i f ica t ion 2: vo lu m e 75 ~l ,MgC12 2.5 mM, inner pr imers SK38 andSK39 (HPLC-pur i f ied, Operon) 75pmol es each , Ampl i T aq 1 .03 un i t ( add i -t i o n a l ) , d e o x y n u c l e o s i d e t r i p h o s p h a t e s200 [J,M ( add i t i ona l ) .

M a s t er m i x f o r a m p l i f i c a t i o n 1 w a sp r e p a r e d a n d a l i q u o t e d i n t o 0 . 5 - m l G e n eAmp r eac t i on t ub es (P e rk i n -E l m er Ce t us )a t 10 .2 i ~l pe r t ube . F our d rops o f mi n e ra lo i l w e r e i n t r o d u c e d i n t o e a c h t u b e , a n dt hen 40 t zl o f DNA prep ara t i on wasa d d e d t o t h e a q u e o u s u n d e r p h a s e . M a s -t e r mi x fo r r eac t i on 2 was p r epa red ande x p e r i m e n t s f r o m t h i s p o i n t f o l l o w e do n e o f t w o v a r i at i o n s , e a c h b a s e d o n u s eof 25 ~ l o f mas t e r m i x 2 fo r ampl i f i ca -t i on 2 .

Control O pen-tube ProcedureAf t e r P CR ampl i f i ca t i on 1 , t ubes wereo p e n e d w i t h f l a m e d f o r c e p s , a q u e o u smas t e r mi x 2 ( s t o r ed a t 4~ was i n t ro -d u c e d i n t o t h e a q u e o u s l a y e r , a n d t h et ubes were r e sea l ed fo r ampl i f i ca t i on 2 .

Closed-tube Procedure with Mas ter Mix 2Sequestered n Agarose Gel Matr ixBefore sea l i ng t he r e ac t i on t ubes fo r am-p l i f i ca t i on 1 , mas t e r mi x 2 i n me l t ed0 .25% agarose (S eakem) a t 70080~ wasa l i q u o t e d w i t h a p o s i t i v e - d i s p l a c e m e n tp i p e tt e i n t o t h e l i d -l o c k c h a m b e r o f t h er eac t i on t ube . T h i s mas t e r m i x was p r e-pa red f rom s t ock a l i quo t s o f 0 .375% aga-

rose i n 1 .5x P CR buf f e r , and s t o r ed a t-2 0~ i n 1 .5 -ml sc r ew-cap t ubes . F ore a c h e x p e r i m e n t , a n a l i q u o t o f b u ff e r /g e li n t h e s t o r a ge t u b e w a s m e l t e d b y b o i l i n g3 - 5 r a i n . W h e n t h e t u b e r e a c h e d7 0 - 80 ~ i t w a s r e m o v e d f r o m t h e w a t e rb a t h a n d t h e o t h e r c o m p o n e n t s o f m a s -t e r m i x 2 w e r e a d d e d a t r o o m t e m p e r a -t u r e d i r ec t l y to t he m e l t ed ge l / buf f e r .T h e c o n t e n t s w e r e m i x e d , r e i n c u b a t e d2-4 m i n a t 70080~ i n t he r e sea l edsc rew-cap t ubes , and t hen a l i quo t ed i n t or e a c t i o n - tu b e l id s a t r o o m t e m p e r a t u r e .A sma l l d rop o f mi ne ra l o i l ( abou t 15 iz l)was l aye red ov er t he so l i d i f i ed ge l . M i n-e r a l o i l was added t o each t ube t o j us tu n d e r t h e l i d- l o ck l in e a n d t h e t u b e w a ssea l ed , l eav i ng a sma l l a i r space i n t he l i d -l o c k c h a m b e r b e l o w t h e a d h e r e n t g e l .

T he e f f ec t o f coo l i ng o n p rese rv i ngm a s t e r m i x 2 i n t h e g e l m a t r i x d u r i n ga m p l i f i c a t i o n I w a s e x a m i n e d b y p l a c i n ga f l a t - b o t to m e d a l u m i n u m b l o c k fi l le dwi t h i ce f l ush over t he r eac t i on t ubes i nt he cyc l e r , whi ch was covered wi t h a r e -p l acement l i d . I ce was r ep l aced eve ry 30m i n , d u r i n g t h e 55 ~ p l a t e a u o f t h e c y-c l e. S i nce the ac t ua l ex t en t o f coo l i n ga c h i e v e d w a s i n d e t e r m i n a t e , t h i s p a r a m -e t er w a s e x a m i n e d m o r e e x a c t l y b y m o c kc l osed- t ube nes t ed P CR. S i mi l a r l y p re -p a r e d r e a c t i o n t u b e s , c o n t a i n i n g m a s t e rm i x 2 g e l i n t h e l i d - l o c k c h a m b e r a n df i ll e d as d e s c r i b e d a b o v e w i t h m i n e r a lo i l, were s to r ed a t 4~ T he n t he l idsw e r e e x c h a n g e d w i t h t h o s e o f e x p e ri -ment a l r eac t i on t ubes a f t e r ampl i f i ca -t i on 1 .

In a l l cases t he ge l con t a i n i ng mas t e rm i x 2 w a s i n t r o d u c e d i n t o t h e r e a c t i o nm i x t u r e b y b r i e f c e n t ri f u g a t i o n a t 1 2 ,5 0 0 g (10-60 sec ) . T ubes were t hen i n -v e r t e d t o m i x t h e c o n t e n t s , r e c e n t r i f u g e das above , and sub j ec t ed t o t he seconda m p l i f i c a t i o n .

F or each am pl i f i ca t i on , t ubes werep l aced i n t he b l ock o f a P e rk i n -E l mer Ce-t us t he r ma l cyc l e r s e t o f 94~ i ncu ba t e da t 95~ fo r 60 sec , and t h en su b j ec t ed t o35 the rm al cycles (95~ 60 sec,55~ x 60 sec, 60~ 60 sec) , fol lo we dby an ex t ens i on s t ep (60~ x 10 mi n) .

U n n e s t e d c o n t r o l a m p l i f i c a t i o n s w e r ea l so pe r fo rm ed w i t h S K380 and S K390 orS K38 and S K39 as p r i mer s . Reac t i on m i xparamet e r s were a s g i ven above fo r am-p l i f i c a ti o n 1 , e x c ep t t h a t p r i m e r c o n t e n twas 50 pm ol es each pe r 50 F ~l o f f i na lr e a c t i o n v o l u m e . P r o v i r u s i n p u t r a n g e df rom 0 t o 100 cop i es pe r t ube i n P BM CDNA and 1000 cop i es i n P CR buf f e r . Am-

p l i f i c a ti o n s f o l lo w e d t h e p r o t o c o l g i v e nabove fo r 35 cyc les and / or 70 cyc les .

Analysis of PCR ProductP CR a m p l i f i c a t i o n p r o d u c t s w e r e a n a -l y z e d b y l i q u i d h y b r i d i z a t i o n w i t h 32pend- l a be l ed S K19 probe . (4~ An a l i quo t o f5 p , l o f each spec i men was hybr i d i zedwi t h 10 0 ,000o115 ,000 c pm of SK19probe i n a f i na l vo l um e of 9 -1 0 i~1 and a1 5 0 mM N aC I . T h e h y b r i d i z a t i o n m i x t u r ew a s d i l u t e d w i t h a n e q u a l v o l u m e o f 2 xs t op b u f f e r a n d t h e e n t i r e c o n t e n t s w e r esubjected to PAGE in 8% gels . X-ray f i lm(Kodak XAR-2) was exposed t o com-p l e t ed ge l s i n cas se t t e s equ i pped wi t h i n -t e n s i f y i n g sc r e e n s fo r 4 - 1 6 h r a t r o o mt e m p e r a t u r e , o r a t - 7 0 ~ f o r m a x i m u msens i t i v i t y .

T o es t i ma t e S K38/ 39 ampl i f i ca t i onp r o d u c t y i e l d i n w e a k t o m o d e r a t e p o s i-t i v e s , s i g n a l o n a u t o r a d i o g r a p h s w a sc o m p a r e d t o t h a t f r o m s e r ia l d i l u t i o n s o fa nes t ed s t rong pos i t i ve (1 -2 I~g p roduc t )o n a u t o r a d i o g r a p h s g i v e n e q u a l e x p o -sure . T he s t rong pos i t i ve was d i l u t ed i nPBMC D NA (0.5 I~g pe r 5 0 pA of PCRbuf fe r ) 3 . 7 x , 1 1 33 1 0 0 a n d t h e n10- fo ld se r i a l l y up t o 1 x 10~ 21 5U n d i -l u t e d s p e c i m e n a n d d i l u t i o n s t h e r e o fw e r e h y b r i d i z e d w i t h t h e s t a n d a r da m o u n t o f S K 1 9 r a d i o l a b e l f o r a u t o r a d -i ography . I t was foun d t ha t : ( 1) t he r e wasa 3.7 to 11 excess of pro du ct for com-p l e te u p t a k e o f p r o b e a n d ( 2) p r o d u c tw a s d e t e c t a b le d o w n to 1 0 - s c o n c e n -t ra t ion ( i .e . , a t 1-2 10 -s I~g).

The SK38/39 ga g a m p l i f i c a t i o n p r o d -uc t o f 115 bp was a l so de t ec t ed d i r ec t l yby aga rose ge l e l ec t rophores i s and e t h i d -i u m b r o m i d e f l u o r e s c e n c e . A n a l i q u o t o18 pA of spec i me n i n 1 s t op bu f f e r wasrun i n a 1% S eakem, 2% Nus i eve ge l ( 1 xTr is-borate-EDTA, pH 8.3) a t 5 V / cm fo rabout 2 h r . A ~ X174 HaeIII DNA di gesw a s u s e d f o r m o l e c u l a r w e i g h t m a r k e r sF o r a s e m i q u a n t i t a t i v e e s t i m a t e o f a m p l i -f icat io n pro duc t yield, 10 !~1 of a 5 di -l u t i o n o f e a c h p o s i t i v e s p e c i m e n i n 1 s t op buf f e r was run s i mi l a r l y aga i ns t s er i a l d i l u t i ons o f t he ~ bX174 HaeIII DNAd i g e st at k n o w n c o n c e n t r a t i o n s . B a n ds t r e n g t h o f d i l u t e d a m p l i f i c a t i o n p r o d -u c t w a s c o m p a r e d t o t h i s s t a n d a r d a n db r a c k e t e d b e t w e e n t h e n e a r e s t g r e a t e ra n d l e ss e r d i l u t i o n s . T h is m e t h o d p r o v e dcapab l e o f d e t ec t i ng / > 1 / 20 p ,g o f S K38/39a m p l i f i c a t i o n p r o d u c t .

M o d i f i e d S o u t h e r n b l o t s w e re p r ep a r e d f r o m a m p l i f i e d m a t e r i a l ru n a s

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a bove a t 10 ~ l pe r s pe c ime n a nd t r a ns -fe r re d by c a p i l l a ry a c t ion w i th 0 .4 NNa OH f ro m the a ga ros e ge l on to a ny lo nme mb ran e (6~ (Zeta-Probe) . The mem -bra ne wa s r in s e d w i th 6x SSC a nd i r ra -d i a t e d wh i l e mo i s t w i th 0 .36 J ou le s / c m 2of UV l ight (Stra ta l inker) . Al l s teps of thep roc e dure de s c r ibe d be low up to the l a s twa s h we re pe r fo rme d a t room t e mpe ra -tu re w i th the hybr id i z a t ion mix tu re un -de r c ons t a n t a g i t a t ion on a p l a t fo rmshaker se t a t 80 cycles /min. The mem-bra ne wa s p re hybr id i z e d in 75 ml o f 6 SSC + 1% SDS for 1 hr , the n hybri dizedi n 1 0 m l o f t h e s a m e m e d i u m c o n t a i n i n gabou t 1 x 106 cpm of ~2p end-labe le dSK19 probe (4~ for 1 hr . The m em br an ewas washed twice wi th 75 ml of 6 SSCfo r 15 min . The wa s h ing wa s c on t inue das above with 2 SSC, 0 .67 SSC, and2x SSC + 1% SDS. A f ina l wash s tep wasdone w ith 2x SSC + 1% SSC a t 37~XAR-2 f i lm was exposed to t he mem -brane in a f i lm casse t te for 2 hr a t roomte mpe ra tu re .

RESULTSDNA s ta nda rds we re f i r st c he c ke d b y theope n- tube ne s t e d PC R c on t ro l p roc e -du re . L iqu id hybr id i z a t ion w i th ra d io l a -be le d SK19 p robe a nd a u to ra d iog ra phydetec ted 32 s t rong pos i t ives (Fig . 1) and3 weak pos i t ives (Fig . 2) . The weakpos i t ives were a l l f rom s ingle-copy pro-virus spec imens . Al l expected negat ivesshowed no de tec table s ignal a f te r f i lme xpos u re up to 16 h r a t -70~ The s eresul ts a re wi th in the expected va lues a te a c h p rov i rus c opy numbe r t e s t e d , f rom33 to zero per tube (Table 1, Series 1). Inpa r t i c u la r , w i th lowe s t -c opy-numbe rpos i t ives , the frac t ion of pos i t ives / to ta lt e s t e d de c re a s e d in a ma nne r c ons i s t e n tw i th the c umula t ive Po i s s on d i s t r ibu t ionfor provirus per spec imen. At 1 .2 copiesper tube , 13 pos i t ives were observed per16 spec imens (95% confidence in terva l ,5 .8-14.1 pos i t ives /16 to ta l ) ; a t 0 .4 copiesper tube , 3 pos i t ives were observed per12 s pe c ime ns (95% c onf ide nc e in t e rva l ,0 .5-7 .5 pos i t ives /12 to ta l ) . These resul tss ugge st t ha t t he ne s t e d PC R p roc e durepicks up a s ingle copy of ta rge t DNA atne a r - the o re t i c a l f r e que nc y . The y i e ld o fSK38 /39 a mpl i f i c a t ion p roduc t i n s t rongpos i t ives was suff ic ient to take up a l lp robe on l i qu id hybr id i z a t ion a nd to a l -low i ts d i rec t de tec t ion by agarose ge le l e c t rophore s i s a nd e th id ium b romidefluorescence (Fig . 3) . A yie ld o f 1-2 ~g

FIGURE 1 Autoradiographic detectio n ofSK38/39 amplification product after hybrid-ization to radiolabeled SK19 probe and PAGEusing closed-tube nested procedure and am-bient cycler temperature. Film exposure wasfor 4 hr at room temperature. Provirus copynum ber of duplicate specimens (underlined)is indicated above the lanes. Fast-migratingprobe is not taken up by negative specimens(lanes 1, 2, and 4). Strong positives from 1.0 to33 copies of provirus take up probe com-pletely, forming two m ajor hybrid bands andone or more weaker bands (lanes 3-12, ar-rows). These represent heteroduplexes, tri-plexes, quadriplexes, etc. A mod erate positivefrom a single-copy specimen takes up probepartially (lane 13) . Titration experimentsbracket the amplification product concentra-tion in this and another nested single-copymoderate positive between 1 and 3% that ofstrong positives.

p roduc t pe r s pe c ime n wa s e s t ima te d bythis procedure , represent ing an ampli f i -ca t ion eff ic iency of 5-10% based on 16.7~g equiva lent of DNA per 200 ~M deox-ynuc leos id e t r iphosp hates in 75 ~l of re -a c t ion mix tu re . The re wa s no obv iousg ra d ie n t o f p roduc t y i e ld f rom s ing le -c opy to mu l t ip l e -c opy s t rong pos i t i ve s .B y a u to ra d iog ra ph ic t i t r a t ion e xpe r i -ments , the y ie ld of SK38/39 ampli f ica-t ion product in a l l three weak pos i t iveswa s b ra c ke te d be twe e n 10 - 4 a nd10 -~ x tha t o f s t rong pos it i ves . Thecause of these nes t ing fa i lures is notc lear , but s ignal s t rength on autoradiog-raphy was in the range of s ingle-copypos i ti ve s de t e c t e d by unne s t e d a mpl i f i-ca t ion w i th SK38 and 39 pr imers (see be-low). By ge l / f luorescence , v i r tua l ly a l ls pe c ime ns , i nc lud ing the s e , s howe d the

us ua l pa t t e rn o f nons pe c i f i c ba nds , i nd i -c a t ing tha t a mpl i f i c a t ion 1 p roc e e de dnorm a l ly ( the one e xc e p t ion i s d i s c uss e dbelow).

T h e e f f ec t o f i n d e t e r m i n a t e a m o u n t so f r e s idua l e nz yme a nd de o xynuc le os idet r iphos pha te s f rom a mpl i f i c a t ion 1 onampli f ica t ion 2 as se t up here wasc he c ke d by runn ing a c onve n t iona l p ro -cedure wi th the same ser ies of dupl ica teDNA s tandards . Here a 12.5-~1 a l iqu ot ofc omple t e d a mpl i f i c a t ion 1 re a c t ion mix -ture (SK380 and 390 primers a t 50pmoles each in 50 ~l) was t rans ferred toa ne w re a c t ion tube fo r a mpl i f i c a t ion 2(SK38 and 39 pr imers a t 75 pmoles eachin 75 ~l) . In th is manner , enzyme (1 .13un i t s pe r t ube ) a nd de oxynuc le os idetr iph osph ates (200 ~M each) were c lose lycontrol led . Similar y ie lds of SK38/39 am-p l i f i c a t ion p roduc t we re found f rom th i smod if ica t ion , es t im ated a t 1-2 F~g perpos i t ive in the ent i re range tes ted , f rom1 to 1000 provirus copies (da ta no ts hown) .

The ide nt i ty of the 115-bp SK38/39a mpl i f i c a t ion p roduc t i n s e l e c t e d s pe c i -me ns f rom the ne s t e d p roc e dure wa sc onf i rme d on Sou the rn b lo t s , whe re i twas spec if ica l ly and s t rongly visua l izedwith radiolabeled SK19 probe (Figs . 4Ca nd 5C ) a nd wa s ind i s t ingu i s ha b le f romp r o d u c t o f th e u n n e s t e d a m p l i f i ca t i o n 2with SK38 and 39 pr imers (Figs . 4B and58).The c lo s e d - tube ne s t e d p roc e dures howe d s e ns i t i v i ty a pp roa c h ing tha t o fthe open-tube control (Table 1 , Series 2 ;Figs 1 , 2 , and 3) . With one except ion,obs e rve d pos i t i ve s we re w i th in the e x -pe c te d ra nge fo r t he c um ula t ive Po i s s ond i s t r ibu t ion a t t he c a l c u la t e d c opy num-ber of provirus (13 pos i t ives /14 s ingle-c opy spe c ime ns ru n a t a mbie n t c yc le rt e mpe ra tu re ; 95% c onf ide nc e in t e rval ,4 .8--12.7 pos i t ives /14 to ta l ) . Autoradiog-ra phy s howe d 68 s t rong pos i t i ve s a nd 3mode ra t e pos i t i ve s . The s t rong pos i t i ve swere readi ly de tec table by ge l / f luores -c e nc e bu t t he mode ra t e pos i t i ve s we remis s e d by th is t e c hn ique . Two o f t he s es t e mme d f rom s ing le -c opy s pe c ime ns .P roduc t y i e ld o f e a c h wa s b ra c ke te d be -twe e n 1 a nd 3% tha t o f s t rong pos i t i ve sby a u to ra d iog ra ph ic t i t r a t ion . The th i rdmode ra t e pos i t i ve wa s f rom a n 11 -c opys pe c ime n , p roduc t y i e ld e s t ima te d a t3 - 9 % , w h i c h s h o w e d a n a n o m a l o u s p a t -t e rn o f ba nds b y ge l / f luo re s c e nc e (da t ano t s hown) . Among s t rong pos i t i ve s ,SK38/39 product y ie ld was typica l ly in

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FIGURE 2 Autoradiographic detection of SK38/39 amp lif ication product hybridized to SK19probe. Unnested amplif ication for 70 cycles (from left, lanes 1-16) and representative nestedamplif ications ( lanes 17-20) . Film exposure was for 16 hr at room temperature. Provirus copynum ber of duplicate specim ens is given above lanes. See Fig. 1 legend for further detai ls . In theunnested amplif ication, SK38/39 product is weakly detectable down to the single-copy level(arrows). The signal from lane 6 was judged indeterminate and was not improved above back-ground after 16 hr of f i lm exposure at - 70~ (data not shown). A weak single-copy posit ive fromthe open- tube nes ted procedure i s shown in lane 18. The SK38/39 product concentrat ion in thisand al l other weak single-copy posit ives from the nested and unnested procedures was est imatedat 10-4 x to 10 -3x that of strong positives. A modera te posit ive from the closed-tube nestedprocedure, from a specimen with 11 copies of provirus which gave anomalous bands on agarosegel electrophoresis , is shown in lane 19. Estimated product is between 3 and 9% that of strongpositives. Lane 20 shows a strong single-copy posit ive from the nested procedure for compariso n.

t h e r a n g e a c h i e v e d b y t h e o p e n - t u b ec o n t r o l m e t h o d , i .e ., 1 - 2 ~ g p e r t u b e .T h e o p e n - t u b e p r o c e d u r e , h o w e v e r , i n -v a r i a b l y g a v e e q u a l o r s o m e w h a t g r e a t e r

y i e l d s w i t h i n t h e b r a c k e t e d v a l u e s a sj u d g e d b y b a n d s t r e n g t h .

T h e r e w a s a t e n d e n c y t o w a r d f o r m a -t i o n o f a p p a r e n t p r i m e r d i m e r s i n a m p l i -

TABLE 1 C l o s e d -t u b e N e s t e d P C R - - S u m m a r y o f R e su l tsProvirus a Series 1b Series 2 b(copies per Open-tu be open-tu be Agarose gel matrix atreaction) control control 4~ storage cool amb ient tempe rature

0 0/10 0/6 0/6 0/6 0/60.4 3/121.0 11/13 8/11 (1 + +)d 6/12 13/14 c (1 + + )1.2 13/16 (3 + )d3.7 10/10 6/6 6/6 4/4 6/6

11 5/5 5/5 5/5 4/4 (1 + + ) 6/ 633 4/4 5/5 4/4 4/4 5/5aThe standard deviation for provirus cop y num ber (based on 8E5 cell count) is + 13%.b'cposit ives/ total . In each case except c, the fraction of observed posit ives fal ls within the 95%confidence interval for expected posit ives, based on the cumulative Poisson distr ibution ofprovirus per reac t ion volume a t the ca lcu la ted me an copy number .dSignal strength of posit ives detectable only by autoradio graphy is given as + (weak) and + +(moderate) . All other posit ives were strong ( + + + + ) , i .e . , detectable by gel electrophoresis andethidium bromide fluorescence as well as by autoradiography.

FIGURE 3 Agarose gel electrophoresis ofSK38/39 amplif ication product from nestedPCR, direct visualization by ethidium bro-mide fluorescence, negative image. (A) Open-tube control procedure: (M) ~X174 HaelII m o-lecular -weight markers ; (C1 ) unampl i f iedgenome DNA cont ro l, co nta in ing reagents foramplif ica tion 1 and 2; (C2) ampli f ication 1control; (0) zero copies provirus; (1) 1 copy;(3) 3.7 copies; (11) 11 copies. Duplicate spec-imens are bracketed. (B) Open-tube controlprocedure continued: ( lane 1) molecular-weight markers as A; (33) 33 copies. Mockclosed-tube procedure, master mix 2 stored at4~ provirus copy num ber as above. (C)Closed-tube procedure, m aster mix 2 at cyclerambient t empera ture dur ing ampl i f ica t ion 1provi rus copy number and molecular -weightmarkers as A. The arrow at the left of each gelindicates the prominent SK38/39 amplif ica-t ion prod uct of 115 bp, runn ing sl ightly fasterthan the 118-bp marker band in the ~X174HaeIII digest . A modera te single-copy posit ivenot de tec ted by th i s method i s shown in Blane 6 from left. The ar rowhead by lane 3from left (A) ind ica tes the pos i t ion of theSK380/390 amplif ication product of 216 bp,which runs wi th the weak nonspec i f ic bandvisible in most lanes but which i tself is notvisible at these low-copy levels.

f i c a t i o n 2 o f t h e c l o s e d - t u b e p r o c e d u r ew i t h n e g a t i v e o r l o w - c o p y - n u m b e r s p ec -i m e n s ( Fi g. 3) . O c c a s i o n a l w e a k b a n d s o ft h i s s iz e w e r e a l s o s e e n w i t h h i g h e r -c o p y - n u m b e r s p e c i m e n s , i n t h e p r e s en c eo f a p r o m i n e n t S K 3 8 / 39 a m p l i f i c a t i o np r o d u c t ( d a t a n o t s h o w n ) . T h e s e r e s u l t s




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FIGURE 4 Agarose gel electro phore sis of a m-plification product from nested proce dureand unnested amplifications 1 and 2, directvisualization by ethidium bromide fluores-cence, negative image. Molecular-weightmarkers in each outer lane are as described inFig. 3 legend. (A) Amplification 1, 70 cycles.Duplicate specimens at 0, 100, and 1000 cop-ies of provirus were run as indicated by un-derlined numb ers above gel lanes. The 216-bpSK380/390 amplification produ ct (arrowhead)is clearly detectable on ly in the 1000-copyspecimens, amp lified withou t no rmal ge-nom ic DNA background. This overlaps a non -specific band, which is visible in lower-copy-num ber specimens. (B) Amp lification 2, 70cycles. Duplicate spec imens at 0, 100 , and1000 copies of provirus. The 115-bp SK38/39amplification product (arrowhead), yield 1/3-2/3 ~g per specim en, is clearly detectable on lyin the 1000-copy specimens, a mplified with-out normal genomic DNA background. Thisruns s l ight ly behind the nonspeci f ic bandseen in lower-copy-number specimens. (C)Open-tube nested PCR control . Duplicatespecimens at 0, 1.0 (1 positive, 1 negative),and 33 copies of provirus. Arrowhead indi-cates SK38/39 produ ct b an d in positive speci-mens, yield 1-2 ~g each. (D) Closed-tubenested PCR, amb ient cycler temperature. Du-plicate specimens at 0, 1.0 (1 positive and 1negative), and 33 copies of provirus. Arrow-head indicates SK38/39 product ban d in pos-i t ive specimens, yield 1/2-1 ~g in single-copypositive, 1-2 ~g in 33 copy specimens.

s u g g e s t s o m e c o m p e t i t i v e p r i m e r d i m e rf o r m a t i o n i n th e m e t h o d a s p e r f o rm e d .M a s t e r m i x 2 s u b j e c t e d t o a m b i e n t c y c l e rt e m p e r a t u r e i n g e n e r a l y i e l d e d a s m u c hS K 38 /3 9 a m p l i f i c a t i o n p r o d u c t a s m i xcoo l ed o r r e f r i ge r a t ed dur i ng t he f i r s t

FIGURE 5 Mod ified Southern blot from agar-ose gel run shown in Fig. 4. Autoradiographyfor 2 hr at roo m tem perature (see Fig. 4 legendfor furthe r details). Radiolabe led SK19 probeconfirms identity of 216-bp SK380/390 and115-bp SK38/39 am plifica tion products. Som esignal is also seen in th e SK38/39 positives atthe approximate posit ion of single-strandedproduct chains. Weak signal is detectable in100-copy specimens am plified by the un-nested procedure w ith SK38 and 39 primers.The several distinct nonspe cific products pro-duced by each amplification and visualizedon gel/fluorescence in Figs. 3 and 4 are notdetected by SK19 probe.

a m p l i f i c a t i o n o f t h e c l o s e d -t u b e p r o c e -dure , bu t t he y i e l d o f occas i ona l spec i -m e n s w a s s o m e w h a t l o w e r (0 . 5 -1 ~ g p ers t rong posi t ive; see F ig. 4D) . Al l s t rongpos i t i ves f rom t he c l osed- t ube p rocedureus i ng coo l i ng o f mas t e r mi x 2 o r r e f r i g -erat ion gave yields in the 1- to 2-p.gr ange . T hese r e su l t s suggest t ha t s ens i t i v -i t y o f t he c l osed- t ube nes t ed p rocedurem a y b e e n h a n c e d s o m e w h a t b y c o o l in go f m a s t e r m i x 2 d u r i n g a m p l i f i c a t i o n 1 .W h e n e x a m i n e d o n S o u t h e rn b l o ts , t h e115-bp S K38/ 39 amp l i f i ca t i o n p rod uc tf r o m t h e c l o s e d - t u b e n e s t e d p r o c e d u r eshowed t he expec t ed cha rac t e r i s t i c s (F i g .5D) .

T h e a b i l i t y o f t h e u n n e s t e d c o m p o -ne n t a mpl i f i ca t i on s 1 and 2 t o de t ec t ta r -g e t p r o v ir a l D N A w a s c o m p a r e d w i t ht h a t o f t h e n e s t e d a m p l i f i c a t i o n pr o c e-d u r e . U n n e s t e d a m p l i f i c a t i o n 1 w i t hS K380 and 390 pr i m er s fo r 70 cyc l es de -t ec t ed d own t o 11 t o 3 .7 cop i es o f p rov i -r u s w i t h w e a k s i g n a l o n a u t o r a d i o g r a p h y

( d a ta n o w s h o w n ) . T h e p r e d i c t e d 2 1 6 - b pS K 3 80 / 39 0 a m p l i f i c a t i o n p r o d u c t w a s v i-s u a l i z e d o n l y f r o m 1 0 0 0 - p r o v i r u s - c o p ys p e c i m e n s o n g e l e le c t r o p h o r e s i s a n dSo uth ern blots (F igs . 4A an d 5A) . Un-n e s t e d a m p l i f i c a t i o n 2 w i t h S K 38 a n d 3 9p r i m e r s , w h e t h e r r u n f o r 35 c y c l es (3posi t ives /4 total ) or for 70 cycles (3 pos-i t ives , 1 i nd e t e r mi n a t e / 4 t o t a l ) , de t ec t edd o w n t o a s i n g l e c o p y o f p r o v i ru s w i t hw e a k t o i n d e t e r m i n a t e s i g n a l o n a u t o r a-d i ography (F i g . 2 ) . T here was no s i gn i f i -c a n t i m p r o v e m e n t i n s i g n a l s t r e n g t hg a i n e d b y t h e a d d i t i o n a l c y c l i n g , e x c e p tfo r 1000-copy spec i mens , where a s t r on-ge r 115-bp S K38/ 39 p roduc t band was v i -s u a l i z e d b y g e l / f l u o r e s c e n c e a n d S o u t h -ern blot t ing (F igs . 4B and 5B) .

C o m p a r a t i v e e f f i c i e n c i e s o f a m p l i -f i c a t io n o f t h e n e s t e d a n d u n n e s t e dp r o c e d u r e s w e r e e s t i m a t e d b y t h r e e a p -proaches . F i r s t, ba nd s t r eng t h s o f amp l i -f i c a t i o n p r o d u c t s f r o m k n o w n c o p y -n u m b e r s p e c i m e n s w er e c o m p a r e d o ne t h i d i u m b r o m i d e - s t a i n e d a g a r o s e g e l sa n d o n d e r i v a t i v e S o u t h e r n b l o t s . S K 3 8 /3 9 a m p l i f i c a t i o n p r o d u c t w a s s t r o n g l yv i s i b l e i n t h e n e s t e d s i n g l e - c o p y p o si -t i ves (1 / 7 .5 o f spec i men run) , bu t t h i sp r o d u c t a n d t h e S K 3 80 / 39 0 p r o d u c t w a sf o u n d a t e q u i v a l e n t t o s o m e w h a t l e s se rc o n c e n t r a ti o n s o n l y i n t h e u n n e s t e d1 0 0 0 - c o p y s p e c i m e n s ( 1 / 5 o f s p e c i m e nrun) (F igs . 4 and 5) . The 1000-copy spec-i m e n s w e r e r u n w i t h o u t n o r m a l P B M CDNA as d i l uen t t o v i sua l i ze spec i f i c am-p l i f i c a t io n p r o d u c t b e t t e r a n d t o o b v i a t ep o t e n t ia l i n h i b i t i o n o f a m p l i f i c a t io n o ft he t a rge t p rov i r a l DNA sequences . T heser e s u lt s s u g g es t t h e a m p l i f y i n g p o w e r o ft h e n e s t e d p r o c e d u r e s i s ~ 1 5 0 0 x t h a t o ft h e u n n e s t e d c o m p o n e n t a m p l i f i c a t i o n sI and 2 . S econd , t he a mo un t s o f S K38/39a m p l i f i c a t io n p ro d u c t f r o m n e s t e d a n du n n e s t e d p r o c e d u r e s n e c e s s a r y t o t ak eu p S K 19 p r o b e c o m p l e t e l y o n l i q u i d h y -b r i d i z a t i o n a n d a u t o r a d i o g r a p h y w e r ec o m p a r e d i n ti t r a t io n e x p e r i m e n t s .S K38/39 p rod uc t f rom 70 cyc les o f un-n e s t e d a m p l i f i c a t i o n to o k u p p r o b e c o m -p l e t e l y b e t w e e n 1 0 0 a n d 1 0 0 0 p r o v i r u sc o p i e s p e r s p e c i m e n ( 1 / 10 o f s p e c i m e nrun , F i g . 2 ) , whereas t he nes t ed p roduc tw a s 3 . 7 x t o 1 1 x i n e x ce s s o f t h e a m o u n tn e c e s s a r y t o a c c o m p l i s h t h e s a m e ( 1 /1 5o f s p e c i m e n r u n , d a t a n o t s h o w n ) . T h e s eresu l t s sugges t 600- -16 ,000x grea t e r am-p l i f y i n g p o w e r o f t h e n e s t e d p r o c e d u r e so v e r th e u n n e s t e d . V i s u a l e s t i m a t i o n o ft h e d a t a i s m o s t c o n s i s t e n t w i t h a s e v er a lt h o u s a n d f o l d i n c r e as e . T h i r d , a s di s -

64 PCR Methods and Applications



7/7

cussed above , a nes t ed s t rong pos i t i ve r e -q u i r e d 1 0 0 0 - 1 0 , 0 0 0 x d i l u t i o n t o m a t c ht h e a t t e n u a t e d s i g n a l o f si n g l e - c o p y p o s-i ti v e s f r o m t h e u n n e s t e d a m p l i f i c a t i o n .As fo r modera t e pos i t i ves f rom t hec l osed- t ube nes t ed p rocedure , t i t r a t i one x p e r i m e n t s s h o w e d t h a t t h e s e w e r e a tl e as t 1 0 - 1 0 0 x s t r o n g e r t h a n t h o s e o f t h eu n n e s t e d p r o c e d u r e a t e q u i v a l e n t p ro v i -r u s c o p y n u m b e r .

DISCUSSIONA c l osed- t ube nes t ed P CR proce dure hasb e e n d e v e l o p e d t h a t s h o w s h i g h s e n s i -t i v it y a p p r o a c h i n g t h a t o f th e o p e n - t u b eprocedure . A s i ng l e copy of HIV ga g tar-ge t DNA i s de t ec t ed a t nea r - t heore t i ca lf r e q u e n c y , t y p i c a ll y w i t h m i c r o g r a myi e l ds o f S K38/ 39 ampl i f i ca t i on p roduc t .A l t h o u g h i t h a s b e e n d e v e l o p e d t o d e t e c tHIV ga g s e q u e n c e s , t h is p r o c e d u r e c o u l db e a d a p t e d f o r a m p l i f i c a t i o n o f o t h e r t ar -ge t DNA sequences a s we l l . As des i gnedhere fo r de t ec t i on purposes , t he s i ng l e -t u b e p r o to c o l i n c o r p o r a t e s s o m e i n d e -t e r m i n a t e e x c es s o f re a g e n t s i n a m p l i -f i ca t i on 2 . T h i s r e su l t s i n no l os s o fa m p l i f y i n g po w e r . F o r a m p l i f i c a t i o n a n dmo re exac t ana l ys i s o f t a rge t s equences ,e .g . , by DNA sequenc i ng , i t may be ad-v i s a b l e t o d r o p d e o x y n u c l e o s i d e t r i p h o s -p h a t e i n p u t f o r a m p l i f i c a t i o n 1 s o t h a tt hese do no t exceed 200 p.M for ampl i f i -c a t i o n 2 , o r o t h e r w i s e m o d i f y r e a c t i o np a r a m e t e r s, t o m i n i m i z e p o t e n t i a l i n c o r-pora t i on e r ro r s by Ta q pol ym erase . (7)

T h e u n n e s t e d a m p l i f i c a t io n w i t hS K38 and 39 p r i mer s a l so showed t heab i l i t y t o de t ec t a s i ng l e copy of p rov i rus ,b u t w i t h m a r g i n a l s i g n a l . W h i l e i t s r e -p r o d u c i b i l i t y h a s n o t b e e n s t u d i e d s y s-t e m a t i c a l l y i n t h i s l a b o r a t o ry , u n n e s t e da m p l i f i c a t i o n u s i n g a s o m e w h a t d i ff e r-en t cyc l e r p rog ram (s) p i cked u p 3 .7 -1 .2cop i es o f p rov i rus . T he c l osed - t uben e s t e d p r o c e d u r e t y p i c a l l y a m p l i f i e s t h et a rg e t D N A s e q u e n c e s e v e ra l h u n d r e d -f o ld t o a t h o u s a n d f o l d m o r e t h a n t h eu n n e s t e d p r o c e d u r e w i t h o u t s i g n i f i -c a n t l y i n c r e a s i n g t h e c h a n c e f o r f a l s epos i t ives . Hence , i t confe r s pa r t i cu l a r ad-v a n t a g e s i n d e t e c t i o n a n d a n a l y s i s o fr a re or u n c o m m o n t a rg e t s e q u e n ce s ,w h e r e t h e a b i l i ty o f e v e n a h i g h l y e f fi -c i en t unn es t e d p rocedure , ( 4 's ) adap t edfor use here , i s taxed.

T hi s s t udy i s based on a p ro t o t y pe de -s i gn fo r c l osed- t ube nes t ed P CR wherel i qu i d mas t e r m i x 2 i s r e t a i ned i n t he l i d -l o c k c h a m b e r o f t h e r e a c t io n t u b e w i t h

a n i n e r t m e m b r a n e ( p a r a f i lm , T e fl o nt a p e) a n d c o o l e d d u r i n g a m p l i f i c a t i o n 1 ,t h e n d i s c h a r g e d a s a b o v e f o r a m p l i f i c a -t i on 2 . T he i n i t i a l des i gn ve r i f i ed t hep r i n c i p l e o f t h e a p p r o a c h b u t p r e s e n t e ds i g n i f i c a n t le a k a g e p r o b l e m s . C o n s e -q u e n t l y , t h e p r e s e n t m e t h o d w a s d e v e l -oped fo r be t t e r r e t en t i on and seques t e r -i n g o f m a s t e r m i x 2 d u r i n g a m p l i f i c a t i o n1 . M os t o f t he deadspace o f t he r eac t i ont ube i s f i l l ed wi t h mi n e ra l o i l , l eav i ng asmal l i nsu l a t i ng a i r l ock i n t he l i d - l ockc h a m b e r u n d e r t h e m a s t e r m i x 2 g e l .W i t h t h i s m o d i f i c a t i o n , s e n s i t i v i ty a p-p r o a c h i n g t h a t o f t h e o p e n - t u b e p r o ce -d u r e h a s b e e n a t t a i n e d , e v e n w h e n m a s -t e r m i x 2 i s s u b i e c t e d t o t h e a m b i e n tt e m p e r a t u r e o f t h e c y c l e r d u r i n g a m p l i -f i ca t i on 1 . Occas i ona l pa r t i a l o r com-p l e t e nes t i n g f a i lu r es were obse rved b o t hw i t h t h e o p e n - t u b e c o n t r o l a n d t h ec l o s e d -t u b e m e t h o d . T h e c a u s e o f t h es ef a i l u r e s r e m a i n s t o b e d e t e r m i n e d . O n em a y s p e c u l a t e t h a t t h e w e a k p o s i t i v e sf rom s i ng l e - copy spec i mens r e f l ec t f a i l -u r e o f a m p l i f i c a t i o n 1 d u e t o e p i s o d i c,i n a d e q u a t e u n m a s k i n g o f l o n g e r p r o v i -r a l DNA sequences a f t e r dena t ura t i on o fg e n o m i c D N A . T h e m o d e r a t e p o s i t i v e sg e n e r a t e d b y t h e c l o s e d -t u b e m e t h o ds p e a k f o r i n h e r e n t t e c h n i c a l f a i lu r e s a n da r e b e i n g d e a l t w i t h b y o n g o i n g r e f i n e -m e n t s . A l t h o u g h t h e n u m b e r o f e x p e r i -m e n t s i s l i m i t e d , t h e m o r e c o n s i s t e n t l yh i g h y i e l d s o f S K 3 8 / 3 9 a m p l i f i c a t i o nproduc t w i t h r e f r i ge r a t ed o r coo l ed mas -t e r mi x 2 sugges t t ha t c oo l i ng does i m-prove se ns i t i v i t y t oward t h a t o f t he con-t ro l p rocedure . A cyc l e r i nco rpora t i ng ane f f i c i en t coo l i ng p l a t e f l ush ac ros s t her e a c t i o n t u b e t o p s d u r i n g a m p l i f i c a t i o n1 wou l d o p t i mi z e r e su lt s . T he exper i -m e n t a l d e s i g n t h a t h a s e v o l v e d h e r e i sb a s e d m o r e o n m a t e r i a l s t h a n c o n c e p -t ua l cons t r a i n t s . F or i ns t ance , any num-b e r o f a p p r o a c h e s b a s e d o n t h e s a m ep r i n c i p l e m a y b e v i s u a l i z e d t h a t t r a pm a s t e r m i x 2 i n a n a p p r o p r i a t e l y e n g i -n e e r e d r e a c t i o n t u b e c h a m b e r o r s u i t -ab l e i ne r t ba r r i e r .

ACKNOWLEDGMENTSE ve l yn Ke l l e r o f t he Vi ro l ogy L abora t o ryk i nd l y supp l i ed 8E 5 cel l s used h e re ; L eoG r a d y o f t h e s a m e l a b o r a t o r y f u r n i s h e dspace fo r p r epa ra t i on and s t o r age o f P CRreagen t s . T he ded i ca t ed t yp i ng as s i s -tance of Ms. Margaret Prescot t i s a l so ac-k n o w l e d g e d .

REFERENCES1. M ullis, K.B. and F.A. Faloona. 1987 . Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction. MethodEnzymol. 155: 335-350.2. Higuch i, R. 1989. Rapid, efficient DNA extraction for PCR from cells or blood. Am-

plifications 2: 1-3.3. Gen delm an, H.E., T.S. Theodore , R. Willey et al. 1987. Mole cular character izationof a pol ymerase mut an t hum an i mmun-odeficiency virus. Virology 160: 232-239.4. Ou, C.Y., S. Kwo k, S.W . Mitc hell et al1988. DNA am plific ation for direct detec-t ion o f HIV-1 in DNA of peripheral b loodmononuclear cells. Science 239 : 295-2975. Rogers, M.F., C.Y. Ou, M. R ayfield et al1989. Use of the polymerase chain reaction for the detection of the proviral sequences o f t he human i mmunodef iciency virus in infan ts born to serposit ivemothers. N. Engl. J. Med. 320: 1649-16546. Reed, K.C. and D.A. Mann . 1985. Rapidtransfer of DNA from agarose gels to nylon membranes . Nucle ic Ac ids Res

13: 7207-7221.7. Saiki, R.K. 1989 . The d esign a nd optim i-zation of the PCR. In PCR technology: Principles and applications for DNA amplification (ed. H.A. Erlich), pp. 7-16 . Stock tonPress, New York.

Received January 30, 1992; accepted inrevised form May 11, 1992.



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